It is proposed to continue studies on the biosynthetic pathways of isoleucine and valine in Escherichia coli. Major effort will be devoted to demonstrating in vitro the interaction of regulatory elements identified in vivo with the components of the transcription and translation systems involved in the expression of the ilv genes. The analysis will consist of finding mutants with altered regulation and defining their effects in vivo on message formation or breakdown and on the translation of the message. The physiological perturbations of gene expression will also be defined in terms of the way they effect transcription and translation of the ilv genes. Attempts will be made to define genetically and physically the precise regions in the ilv gene cluster that interact with regulatory elements. Finally, we hope to mimic with in vitro transcription and translation the regulation of the ilv gene cluster as defined in vivo.